ViroBytes is a modified [http://2009.igem.org/Team:Alberta BioBytes] procedure used for "rational shuffling" of capsid genes from several Adeno-Associated Virus serotypes. Main motivation for the new protocol is unsatisfactory incorporation of certain serotypes (and especially certain parts of AAV capsid regions eg. from AAV5) observed in standard shuffling techniques ([http://2010.igem.org/Team:Heidelberg/Project/References#Capsid_Shuffling Grimm et al., 2008]). The principle of BioByte formation and annealing persists. Sticky overhangs are used for selective combination of the bytes but different method is used for the production of individual ViroBytes.

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ViroBytes is a modified [http://2009.igem.org/Team:Alberta BioBytes] procedure used for "rational shuffling" of capsid genes from natural isolates of Adeno-Associated Virus (AAV). Main motivation for the new protocol is unsatisfactory incorporation of certain serotypes (and especially certain parts of AAV capsid regions eg. from AAV5) observed in standard shuffling techniques ([http://2010.igem.org/Team:Heidelberg/Project/References#Capsid_Shuffling Grimm et al., 2008]). The principle of BioByte formation and annealing persists. Sticky overhangs are used for selective combination of the bytes but different method is used for the production of individual ViroBytes.

Five AAV serotypes (1,2,5,6,8 and 9) were selected as suitable candidates for fragmentation due to their exceptional individual properties. The analysis of Cap gene sequences revealed multiple homology regions which were then used for rational fragment formation. Total number of fragments per Cap gene is eight in our case and all fragments have similar length around ~250bp to assure similar behaviour in the ligation reaction. The Bytes are created and amplified by High-Fidelity PCR using Cap- and FragmentX-specific primers. These primers contain flanking regions with recognition sequence for type II restriction enzyme Bsa1. Precise positioning in front of and behind the homology regions at the ends of each fragmetn ensures that the enzyme forms unique sticky ends with 4nt overhangs. This procedure allows the user to avoid arduous design of overhangs with incorporated uracils further used for USER<sup>TM</sup> digestion. Also for the purposes of shuffling, the homology regions need to be exploited in order to avoid frame shifts which cannot be easily accomplished using standard BioByte protocol.

Five AAV serotypes (1,2,5,6,8 and 9) were selected as suitable candidates for fragmentation due to their exceptional individual properties. The analysis of Cap gene sequences revealed multiple homology regions which were then used for rational fragment formation. Total number of fragments per Cap gene is eight in our case and all fragments have similar length around ~250bp to assure similar behaviour in the ligation reaction. The Bytes are created and amplified by High-Fidelity PCR using Cap- and FragmentX-specific primers. These primers contain flanking regions with recognition sequence for type II restriction enzyme Bsa1. Precise positioning in front of and behind the homology regions at the ends of each fragmetn ensures that the enzyme forms unique sticky ends with 4nt overhangs. This procedure allows the user to avoid arduous design of overhangs with incorporated uracils further used for USER<sup>TM</sup> digestion. Also for the purposes of shuffling, the homology regions need to be exploited in order to avoid frame shifts which cannot be easily accomplished using standard BioByte protocol.

ViroBytes

Introduction

ViroBytes is a modified BioBytes procedure used for "rational shuffling" of capsid genes from natural isolates of Adeno-Associated Virus (AAV). Main motivation for the new protocol is unsatisfactory incorporation of certain serotypes (and especially certain parts of AAV capsid regions eg. from AAV5) observed in standard shuffling techniques (Grimm et al., 2008). The principle of BioByte formation and annealing persists. Sticky overhangs are used for selective combination of the bytes but different method is used for the production of individual ViroBytes.

Five AAV serotypes (1,2,5,6,8 and 9) were selected as suitable candidates for fragmentation due to their exceptional individual properties. The analysis of Cap gene sequences revealed multiple homology regions which were then used for rational fragment formation. Total number of fragments per Cap gene is eight in our case and all fragments have similar length around ~250bp to assure similar behaviour in the ligation reaction. The Bytes are created and amplified by High-Fidelity PCR using Cap- and FragmentX-specific primers. These primers contain flanking regions with recognition sequence for type II restriction enzyme Bsa1. Precise positioning in front of and behind the homology regions at the ends of each fragmetn ensures that the enzyme forms unique sticky ends with 4nt overhangs. This procedure allows the user to avoid arduous design of overhangs with incorporated uracils further used for USERTM digestion. Also for the purposes of shuffling, the homology regions need to be exploited in order to avoid frame shifts which cannot be easily accomplished using standard BioByte protocol.